Recent studies showed that clotting factor FVIIa (FVIIa), which initiates the coagulation cascade upon binding to procoagulant cofactor tissue factor (TF) following vascular injury, also binds to anticoagulant cofactor endothelial cell protei C receptor (EPCR). EPCR plays a critical role in the protein C anticoagulant pathway by promoting the activation of protein C. EPCR also promotes activated protein C (APC)-induced cytoprotective signaling. Studies conducted in the last funding cycle revealed that FVIIa binding to EPCR on the endothelium facilitates the transport of FVII/FVIIa from the circulation to extravascular sites and FVIIa bound to EPCR activates the protease-activated receptor 1 (PAR1)-mediated cell signaling and provides the barrier protective effect. Pharmacological concentrations of rFVIIa were found to displace endogenous protein C from EPCR. rFVIIa is routinely used to treat hemophilia patients with inhibitory antibodies against FVIII and FIX. It is also being used as an emergency hemostatic agent in both pediatric and adult patients. Despite its widespread and successful use, the understanding of the mechanism of rFVIIa action in bleeding disorders is incomplete. A clear understanding of the mechanistic action of rFVIIa in therapy is essential to improve the clinical efficacy of rFVIIa, and to better manage patients and develop a new generation of FVIIa-based therapeutics. We hypothesize that pharmacological concentrations of rFVIIa compete with endogenous protein C for the limited EPCR sites on the endothelium, which diminishes EPCR-dependent APC generation, and thus the down-regulation of the APC anticoagulant pathway. The down-regulation of the APC anticoagulant pathway would enhance FVIIa-induced thrombin generation as factor Va is not readily inhibited by the reduced APC levels. We also hypothesize that EPCR-mediated FVIIa transport and FVIIa-EPCR-mediated barrier protective and anti-inflammatory effects play crucial roles in preventing the joint damage and bone loss in rFVIIa prophylaxis. The following aims will test the above hypotheses. Aim 1: Investigate the importance of FVIIa:EPCR interaction in the hemostatic effect of rFVIIa in treating bleeding disorders and elucidate its mode of action; Aim 2: Characterize FVIIa bio-distribution and tissue retention in prophylaxis and evaluate the contribution of FVIIa-EPCR-mediated hemostatic, barrier protective and anti-inflammatory effects to the prophylactic effect of preventing hemophilic arthropathy; and Aim 3: Elucidate the mechanism by which EPCR ligation with EPCR mAb, in the absence of rFVIIa treatment, provides the vascular barrier protective effect in hemophilia. To perform these studies, we will generate novel mFVIIa variants and unique transgenic mice and employ saphenous vein bleeding, the needle puncture knee injury, and VEGF-and LPS- induced vascular leakage model systems. The outcome of the proposed studies will introduce a paradigm shift in our understanding of how the pharmacological concentration of rFVIIa provides the hemostatic effect in hemophilia patients and patient with other bleeding disorders.